Recombinant adenovirus is an appropriate vector for endocytotic protein trafficking studies in cultured neurons

He Yuan, Ping Zhai, Leonard M. Anderson, Jie Pan, Bayar Thimmapaya, Edward H. Koo, Numa R. Marquez-Sterling

Research output: Contribution to journalArticle

10 Scopus citations

Abstract

Endocytosis of full-length β-amyloid precursor protein (APP) from the plasma membrane contributes to β-amyloid peptide (Aβ) secretion, and, hence, potentially contributes to the molecular pathogenesis of Alzheimer's disease. We recently have demonstrated that central neuronal APP is endocytosed in a common vesicular compartment with recycling synaptic vesicle integral membrane proteins, but is then sorted away from synaptic vesicles for retrograde transport to the neuronal soma. For this report, we explore whether recombinant adenovirus can be used to modulate APP expression in cultured central neurons to study APP processing by the endocytotic pathway in these cells. Using a replication-deficient recombinant adenovirus that expresses a lacZ reporter (Ad5/CMV-lacZ), we demonstrate high efficiency of transfection (30-35%) at low viral titer (10-20 MOI), with no significant neuronal toxicity or cytoarchitectural change. In addition, we demonstrate that infection with the control virus does not result in re-direction of endogenous neuronal APP from usual endocytotic pathways. We have prepared, using the same genomic background as the control virus, an adenoviral vector that expresses the neuronal isoform of human APP (Ad5/CMV-APP). Infection with Ad5/CMV-APP at 10-20 MOI results in significantly increased immunoreactivity for endocytosed APP with preservation of usual endocytotic trafficking. These results demonstrate that recombinant adenovirus at low titer is an appropriate and effective vector for protein trafficking/processing studies in cultured central neurons.

Original languageEnglish (US)
Pages (from-to)45-54
Number of pages10
JournalJournal of Neuroscience Methods
Volume88
Issue number1
DOIs
StatePublished - Apr 1 1999

Keywords

  • Alzheimer disease
  • Amyloid precursor
  • LacZ reporter

ASJC Scopus subject areas

  • Neuroscience(all)

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